Aptamers screening method based on graphene without target immobilization and the aptamers obtained from the method

ABSTRACT

Provided is aptamers screening method based on graphene without target immobilization and the aptamers obtained from the method, and more particularly, a new GO-SELEX method without target immobilization in which a single-stranded nucleic acid pool may react with a non-bound target material or a counter-target material, after which a single-stranded nucleic acid which has not been bound to the target or counter-target may be separated by using the graphene. Also, the specific aptamer obtained through the above-described method may be used for diagnosing target related diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 14/342,241, filed Feb. 28, 2014, which is the U.S. National Phase ofInternational Application No. PCT/KR2012/006927 filed Aug. 30, 2012,which claims priority to Korean Patent Application Nos. 10-2012-0043230,filed Apr. 25, 2012 and 10-2011-0088066, filed Aug. 31, 2011. Thedisclosures of all the applications are incorporated herein by referencein their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which 28 has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 28, 2014, isnamed SequenceListing_G16U13C0217P.txt and is 8 KB in size.

BACKGROUND

1. Field of the Invention

The present invention relates to aptamers screening method based ongraphene without target immobilization in which a single-strandednucleic acid pool reacts with a non immobilized target material and asingle-stranded nucleic acid which has not been bound to the targetmaterial or a counter-target material is separated using the graphene,and also relates to specific aptamers selected by the method.

2. Discussion of Related Art

In the conventional biosensor field, an antibody is superior in terms ofsensitivity to other various sensing materials used to detect a targetmaterial. However, antibodies have a problem in that a process ofinjecting a target material (antigen) to be detected into an animal'sbody, obtaining an antibody produced by the animal's biological immunesystem, and performing a purification process is time-consuming andcostly. Further, as compared with aptamers that are rarely limited intarget materials, there is a limit in target materials that can be usedas antigens. Therefore, it is difficult to manufacture antibodies to lowmolecular chemical substances such as toxic substances. Typically, anantibody is a very big protein having a size of 100 KDa or more, andthus, when it is used for an electrochemistry-based biosensor, it may belimited in signal detection, and it is remarkably inferior in terms ofthermostability to DNA or other chemical substances. Therefore, aconventional technique using an antibody for diagnosing a bloodbiomarker is not efficient in terms of cost and time, cannot be appliedto various fields and is limited in application as a biosensor.

As a novel sensing material to solve such problems, an aptamer which isa nucleic acid construct having a specifically high affinity to varioustarget materials has been used in many studies.

An aptamer is a single-stranded DNA or RNA construct having highspecificity and affinity to a specific target. Since an aptamer has ahigh affinity to a target, has an excellent thermostability and can besynthesized in vitro, it is superior in terms of cost to other sensingmaterials used in the conventional sensor field. Further, there is nolimit in a target material, and thus it is possible to synthesizeaptamers with respect to various targets including biomolecules such asproteins, amino acids, etc., small organic chemical substances such asendocrine disruptors or antibiotics, and bacteria. In recent years, dueto characteristics of aptamers specifically bound to target materials,many studies on developing an aptamer for application to new drugdevelopment, a drug delivery system, and biosensors have been conducted.

The most important factor in a method for developing an aptamer is todistinguish DNA (or RNA) which is bound to a target from DNA which isnot bound to the target. To do so, studies on distinguishing DNA bytypically immobilizing a target or immobilizing a DNA random libraryhave been conducted. However, the biggest problem of such animmobilization method is that immobilized yield can be low and it iscostly and time-consuming to analyze the immobilized yield. Further, thepossibility of DNA being directly bound to a separation material(magnetic beads, columns, etc.) used for immobilization cannot beentirely excluded, and the possibility of loss of a DNA pool which mayoccur when the DNA bound to a target immobilized to the separationmaterial is separated again remains as a limit and problem of theimmobilization method. In particular, a low DNA immobilization rateproblem which may occur when a DNA library is immobilized is directlyrelated to loss of a DNA pool which is the biggest loss to be avoidedduring an aptamer development process and thus serves as an upper limit.Moreover, it is difficult to develop aptamers by the immobilizationmethod for heavy metal ions which cannot be immobilized, and thus theimmobilization method may be limited in target selection. However, theabove-described limits can be overcome by immobilization-free developingtechnology of aptamer. Further, since a binding site of a target is notlimited, it is possible to reduce the number of repetitions of aselection process required for development of an aptamer. Therefore, inorder to invent a technique by which an aptamer can be developed througha immobilization-free method, a microelectromechanical system (MEMS),capillary electrophoresis, etc. have been conventionally used, butexpensive equipment, complexity in use of devices, necessity of skilledmanpower, and the like still remain as problems. Meanwhile, graphene isa two-dimensional carbon structure having excellent thermostability,electrical characteristics, and strength and is bound to a base of asingle-stranded DNA by π-stacking, and thus a wide range of studiesusing such characteristics have been conducted.

Adipokines are proteins secreted from adipocytes and tissues and playsan important role in metabolism. One of adipokine, Nampt (Nicotinamidephosphoribosyltransferase) is a visceral fat-derived protein that hasbeen newly found in recent years and is reported as being closelyrelated to type-2 diabetes caused by obesity. Further, Nampt inducesangiogenesis of cancer cells and thus it is an important biomarkerassociated with colorectal cancer, prostate cancer, stomach cancer,breast cancer, etc. and is also associated with various diseases such aspolycystic ovarian syndrome, chronic renal failure, chronic obstructivelung disease, etc.

Meanwhile, bovine viral diarrhea is a disease that causes ulcers ingastrointestinal mucous membranes, diarrhea, respiratory diseases, anddeath in severe cases. A Bovine Viral Diarrhea Virus type 1 causingbovine viral diarrhea is one of the main threats to the livestockindustry. If a pregnant cow is infected, fetal infection occurs highlyfrequently and various disabilities such as still birth and congenitalanomalies occur. A fetus born after being infected at an earlygestational stage may carry the virus throughout its life and serve as asource of new infections. In a route of infection with bovine viraldiarrhea, infection from a persistently infected cow is consideredsignificant. Therefore, in order to effectively conduct diseasesurveillance and quarantine activities against bovine viral diarrhea, itis necessary to separate or detect the virus from bovine serum andtissues by a rapid and accurate test method. In this regard, there isreported a method of detecting whether a tissue of a target animal testspositive or negative for the Bovine Viral Diarrhea Virus using anantibody specific to the Bovine Viral Diarrhea Virus epitope (US2003/0143573). However, conventionally, a method using an antibody fordiagnosis has been reported as time-consuming and costly since it isnecessary to inject a target material (antigen) to be detected into ananimal's body to obtain an antibody produced by the animal's biologicalimmune system, and it is also necessary to perform a purificationprocess. Further, there is a limit in target materials that can be usedas antigens. Therefore, it is difficult to manufacture antibodies totoxic substances or viruses. Furthermore, an antibody is a very bigprotein having a size of 100 KDa or more, and thus, when it is used asan electrochemistry-based biosensor, it may be limited in signaldetection, and it is remarkably inferior in terms of thermostability toDNA or other chemical substances, and thus may be limited in applicationto diagnosis. However, conventionally, there has not been reported anaptamer targeting the Bovine Viral Diarrhea Virus.

The present inventors completed the present invention based on thefindings that when graphene is used in developing an aptamer, it ispossible to develop an aptamer without a process of immobilizing atarget, thereby solving all the problems of the conventional techniquessuch as limits in binding sites of a target, which may occur when thetarget is immobilized, and also possible to develop a DNA aptamerwithout skilled manpower or expensive equipment, and a nucleic acidaptamer capable of firstly diagnosing relevant diseases more accuratelyand rapidly by accurately measuring a concentration of Nampt, which is atarget model, as an important biomarker for type 2 diabetes and cancers,and secondly being specifically bound to a Bovine Viral Diarrhea Virustype 1 only without being bound to other similar substances (BVDV type2, MDBK cell, Bovine Serum albumin, etc.) for diagnosing bovine viraldiarrhea more accurately and rapidly has been developed.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a method fordeveloping a graphene nanomaterial-based aptamer without using aseparate device by means of an immobilization-free method.

Another object of the present invention is to provide a nucleic acidaptamer nanostructure as a sensing material with high sensitivity whichcan be specifically bound to a target such as a specific protein in asmall amount, a low molecular substance, and a virus but is not bound toother biomaterials in a blood sample, using the method for developing agraphene-based aptamer.

Still another object of the present invention is to detect or separate aspecific protein, virus, or the like using the specific aptamer and alsodiagnose diseases related to the specific protein, virus, or the like.

In order to achieve the above-described objects, one aspect of thepresent invention provides a method for producing a nucleic acid aptamerwithout target immobilization comprising: reacting a single-strandednucleic acid pool including a PCR primer region at both ends and any 30to 50 bases at its center with a target material or a counter-targetmaterial and adding graphene; and

separating a target specific nucleic acid aptamer by removing a targetnon-specific single-stranded nucleic acid bound to the graphene, orseparating a target specific nucleic acid aptamer from the graphene bycausing a conformational change by the target material on a targetspecific single-stranded nucleic acid bound to the graphene.

To be more specific, the method for producing a nucleic acid aptamerwithout target immobilization comprises the following steps:

a first step in which a single-stranded nucleic acid pool including aPCR primer region at both ends and any 30 to 50 bases at its center ismixed with a target material or a counter-target material in a buffersolution and these are induced to be bound to each other at normaltemperature;

a second step in which the mixture solution is reacted with graphene toremove a single-stranded nucleic acid which is not bound to the targetmaterial or the counter-target material;

a third step in which a single-stranded nucleic acid specifically boundto the target material and obtained in the above step is amplified byperforming a PCR using the PCR primer region;

a fourth step in which a graphene-based selection process and a counterselection process are repeatedly carried out on the single-strandednucleic acid specifically bound to the target material using the targetmaterial and the counter-target material; and

a fifth step in which a target non-specific single-stranded nucleic acidbound to the counter-target material in the graphene-based selectionprocess is removed, and a target specific aptamer is separated from thegraphene by causing a conformational by the target material on a targetspecific single-stranded nucleic acid bound to the graphene.

Further, the present invention provides a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 4 to 18 in which the nucleic acid aptamer isspecifically bound to a Nampt protein.

Furthermore, the present invention provides a composition for detectinga Nampt protein comprising: a nucleic acid aptamer having a sequenceselected from the group consisting of the base sequences set forth inSEQ ID NOS: 4 to 18 in which the nucleic acid aptamer is specificallybound to a Nampt protein.

Moreover, the present invention provides a composition for diagnosing aNampt related disease comprising: a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 4 to 18 in which the nucleic acid aptamer isspecifically bound to a Nampt protein.

Also, the present invention provides a solid phase carrier immobilizinga nucleic acid aptamer having a sequence selected from the groupconsisting of the base sequences set forth in SEQ ID NOS: 4 to 18 inwhich the nucleic acid aptamer is specifically bound to a Nampt protein.

Further, the present invention provides a kit for detecting a Namptprotein comprising: a nucleic acid aptamer having a sequence selectedfrom the group consisting of the base sequences set forth in SEQ ID NOS:4 to 18 in which the nucleic acid aptamer is specifically bound to aNampt protein.

Furthermore, the present invention provides a kit for diagnosing a Namptrelated disease comprising: a nucleic acid aptamer having a sequenceselected from the group consisting of the base sequences set forth inSEQ ID NOS: 4 to 18 in which the nucleic acid aptamer is specificallybound to a Nampt protein.

Moreover, the present invention provides a composition for separating aNampt protein comprising: a nucleic acid aptamer having a sequenceselected from the group consisting of the base sequences set forth inSEQ ID NOS: 4 to 18 in which the nucleic acid aptamer is specificallybound to a Nampt protein.

Also, the present invention provides a Nampt protein separation methodcomprising: bringing a Nampt protein-containing sample in contact with asolid phase carrier immobilizing a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 4 to 18 in which the nucleic acid aptamer isspecifically bound to a Nampt protein; and eluting the Nampt proteinbound to the solid phase carrier using an eluent.

Further, the present invention provides a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 19 to 28 in which the nucleic acid aptamer isspecifically bound to a Bovine Viral Diarrhea Virus type 1.

Furthermore, the present invention provides a composition for detectinga Bovine Viral Diarrhea Virus type 1 comprising: a nucleic acid aptamerhaving a sequence selected from the group consisting of the basesequences set forth in SEQ ID NOS: 19 to 28 in which the nucleic acidaptamer is specifically bound to a Bovine Viral Diarrhea Virus type 1.

Moreover, the present invention provides a Bovine Viral Diarrhea Virustype 1 detection method comprising: bringing a nucleic acid aptamerhaving a sequence selected from the group consisting of the basesequences set forth in SEQ ID NOS: 19 to 28 in which the nucleic acidaptamer is specifically bound to a Bovine Viral Diarrhea Virus type 1 incontact with a Bovine Viral Diarrhea Virus type 1-containing sample todetect the Bovine Viral Diarrhea Virus type 1.

Also, the present invention provides a composition for diagnosing bovineviral diarrhea comprising: a nucleic acid aptamer having a sequenceselected from the group consisting of the base sequences set forth inSEQ ID NOS: 19 to 28 in which the nucleic acid aptamer is specificallybound to a Bovine Viral Diarrhea Virus type 1.

Further, the present invention provides a solid phase carrierimmobilizing a nucleic acid aptamer having a sequence selected from thegroup consisting of the base sequences set forth in SEQ ID NOS: 19 to 28in which the nucleic acid aptamer is specifically bound to a BovineViral Diarrhea Virus type 1.

Furthermore, the present invention provides a kit for detecting a BovineViral Diarrhea Virus type 1 comprising: a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 19 to 28 in which the nucleic acid aptamer isspecifically bound to a Bovine Viral Diarrhea Virus type 1.

Moreover, the present invention provides a kit for diagnosing bovineviral diarrhea comprising: a nucleic acid aptamer having a sequenceselected from the group consisting of the base sequences set forth inSEQ ID NOS: 19 to 28 in which the nucleic acid aptamer is specificallybound to a Bovine Viral Diarrhea Virus type 1.

Also, the present invention provides a composition for separating aBovine Viral Diarrhea Virus type 1 comprising: a nucleic acid aptamerhaving a sequence selected from the group consisting of the basesequences set forth in SEQ ID NOS: 19 to 28 in which the nucleic acidaptamer is specifically bound to a Bovine Viral Diarrhea Virus type 1.

In addition, the present invention provides a Bovine Viral DiarrheaVirus type 1 separation method comprising: bringing a Bovine ViralDiarrhea Virus type 1-containing sample in contact with a solid phasecarrier immobilizing a nucleic acid aptamer having a sequence selectedfrom the group consisting of the base sequences set forth in SEQ ID NOS:19 to 28 in which the nucleic acid aptamer is specifically bound to aBovine Viral Diarrhea Virus type 1; and eluting the Bovine ViralDiarrhea Virus type 1 bound to the solid phase carrier using an eluent.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the attached drawings, in which:

FIG. 1 provides a schematic diagram of a single-stranded nucleic acidaptamer development method using a graphene oxide (GO-SELEX) accordingto the present invention.

FIG. 2 illustrates an increasing ratio of single-stranded DNA bound to aNampt protein as a target model in a graphene oxide-based aptamerdevelopment process according to the present invention.

FIGS. 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16 and 17 provideschematic diagrams of secondary structures of DNA aptamers obtained byanalyzing base sequences of 15 nucleic acid aptamers bound to a Namptprotein with the web server-based M-fold program according to thepresent invention.

FIG. 18 illustrates a result of analyzing specificity of G35, G40, andG55 aptamers (SEQ ID NOS: 8, 14, and 18) as DNA aptamers bound to aNampt protein to other adipokines and HAS according to the presentinvention.

FIG. 19 illustrates a result of analyzing binding force of G35, G40, andG55 aptamers (SEQ ID NOS: 8, 14, and 18) having a high bindingspecificity to a Nampt protein, according to the present invention.

FIG. 20 is a graph showing a percentage of single-stranded DNA bound toa Bovine Viral Diarrhea Virus type 1 (BVDV type 1) as a target materialin each selection round in a selection process for separating an aptamerspecific to the BVDV type 1.

FIGS. 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 provide schematicdiagrams of secondary structures of aptamers obtained by analyzing basesequences of 10 nucleic acid aptamers specific to a BVDV type 1 with theweb server-based M-fold program according to the present invention.

FIG. 31 illustrates a result of binding specificity of an aptamer B1-11(SEQ ID NO: 23), an aptamer B1-34 (SEQ ID NO: 21), and an aptamer B1-43(SEQ ID NO: 26) as aptamers specific to a BVDV type 1 to the BVDV type 1(BVDV t1) and similar substances (BVDV type 2, CSFV, MDBK, and BSA)according to the present invention.

FIG. 32 is a graph showing affinity of an aptamer B1-11 (SEQ ID NO: 23),an aptamer B1-34 (SEQ ID NO: 21), and an aptamer B1-43 (SEQ ID NO: 26)as aptamers specific to a BVDV type 1 to the BVDV type 1.

FIG. 33 provides a schematic diagram of a detection method using asandwich method according to the present invention.

FIG. 34 illustrates a result of binding force when an aptamer B1-11 (SEQID NO: 23) having the lowest K_(d) among aptamers specific to a BVDVtype 1 is immobilized and an aptamer B1-43 (SEQ ID NO: 26) having thehighest K_(d) is reacted in a second-order reaction.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, exemplary embodiments of the present invention will bedescribed in detail. However, the present invention is not limited tothe embodiments disclosed below, but can be implemented in variousforms. The following embodiments are described in order to enable thoseof ordinary skill in the art to embody and practice the presentinvention.

Although the terms first, second, etc. may be used to describe variouselements, these elements are not limited by these terms. These terms areonly used to distinguish one element from another. For example, a firstelement could be termed a second element, and, similarly, a secondelement could be termed a first element, without departing from thescope of exemplary embodiments. The term “and/or” includes any and allcombinations of one or more of the associated listed items.

It will be understood that when an element is referred to as being“connected” or “coupled” to another element, it can be directlyconnected or coupled to the other element or intervening elements may bepresent. In contrast, when an element is referred to as being “directlyconnected” or “directly coupled” to another element, there are nointervening elements present.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of exemplaryembodiments. The singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises,”“comprising,” “includes,” and/or “including,” when used herein, specifythe presence of stated features, integers, steps, operations, elements,components, and/or groups thereof, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components, and/or groups thereof.

With reference to the appended drawings, exemplary embodiments of thepresent invention will be described in detail below. To aid inunderstanding the present invention, like numbers refer to like elementsthroughout the description of the figures, and the description of thesame elements will be not reiterated.

The main terms used in the detailed description of the present inventionwill be defined as follows.

The term “nucleic acid aptamer” refers to a small single-strandedoligonucleotide that can specifically recognize its target material withhigh affinity. Nucleic acid aptamers are offered as single-stranded DNAor RNA. Therefore, if the nucleic acid is RNA, T in a sequence of thenucleic acid is expressed as U. It is obvious to one of ordinary skillin the art that such a sequence is included in the scope of the presentinvention.

The terms “graphene,” “graphene oxide,” “graphene oxide nanosheet,” and“graphene nanosheet” mean two-dimensional carbon structures and may beused interchangeably throughout the present specification.

The term “sample” refers to a composition that contains or is assumed tocontain a target material, for example, a protein, a low molecularmaterial, a virus, etc., and will be analyzed and may be detected from asample collected from any one or more of, but not limited to, liquid,soil, air, food, waste, animal intestines, and animal tissues. Herein,the liquid may be serum, blood, urine, water, tears, sweat, saliva,lymph, and cerebrospinal fluid. The water may include river water,seawater, lake water, and rain water. The waste may include sewage,waste water, and the like. The animal may include a cow. Further, theanimal tissues may include mucous membranes, skin, cortices, hair,scales, eyes, tongues, cheeks, hooves, beaks, snouts, feet, hands,mouths, nipples, ears, noses, etc.

The term “counter-target material” or “counter target” comprehensivelymeans a material belonging to a family which has a similar structure, asimilar active site, or similar activity to a target or a targetmaterial. For example, if a target material is a Nampt protein,adiponectin, RBP4 (Retinol Binding Protein 4), resistin, vaspin, and HSA(Human Serum Albumin) may be used as a counter-target material. If atarget material is a Bovine Viral Diarrhea Virus type 1, Bovine ViralDiarrhea Virus type 2 (BVDV type 2), CSFV (Classical Swine Fever Virus),MDBK (Mardin-Darby Bovine Kidney cell), or BSA (Bovine Serum Albumin)may be used as a counter-target material.

One aspect of the present invention relates to a method for producing anucleic acid aptamer without target immobilization comprising: reactinga single-stranded nucleic acid pool including a PCR primer region atboth ends and any 30 to 50 bases at its center with a target material ora counter-target material and adding graphene; and separating a targetspecific nucleic acid aptamer by removing a target non-specificsingle-stranded nucleic acid bound to the graphene, or separating atarget specific nucleic acid aptamer from the graphene by causing aconformational change by the target material on a target specificsingle-stranded nucleic acid bound to the graphene.

As a general method for developing an aptamer selectively bound to atarget, a SELEX (Systematic Evolution of Ligand by ExponentialEnrichment) method has been widely used. In the present invention, anucleic acid aptamer specifically bound to a target material isdeveloped using a graphene oxide (GO)-SELEX method modified from thegeneral SELEX method.

The GO-SELEX method used in the present invention is the SELEX methodusing graphene oxide to distinguish a nucleic acid which is bound or notbound to a target or a counter target, and the GO-SELEX method uses afluorescence labeled primer during a nucleic acid amplification step,and thus, during a subsequent PCR product separation (for example,dsDNA→ssDNA) step using PAGE, it is necessary to take out only afluorescent band. In the conventional SELEX methods, a radioactivelabel, capillary electrophoresis, a membrane filter, etc. have beenused, but such methods require great expense and are difficult tohandle. One of the core parts of the SELEX method is to separate anucleic acid which is bound to a target material from a nucleic acidwhich is not bound to the target material. In order to separate them,chromatography, an affinity column, and the like have been used, butthey are expensive and less efficient (R. Stoltenburg et al., 2005, AnalBioanal Chem 383: 83-91). However, the present invention ischaracterized in that since a single-stranded nucleic acid which is notbound to a target material or a counter-target material is removed usinggraphene, even if the target material or the counter-target material isnot specifically immobilized to a specific carrier, it is possible toseparate a single-stranded nucleic acid which is bound to the targetmaterial or the counter-target material from the single-stranded nucleicacid which is not bound to the target material or the counter-targetmaterial. By this method, it is possible to simply and easily solve theproblems of the conventional target immobilizing methods. The presentinvention can employ the typical target immobilizing SELEX method as itis except that a single-stranded nucleic acid which is not bound to atarget material or a counter-target material is removed using graphene.

The method for producing nucleic acid aptamer without targetimmobilization of the present invention may further comprise amplifyinga single-stranded nucleic acid specifically bound to the target materialby performing a PCR using the PCR primer region on the single-strandednucleic acid separated from the target material/single-stranded nucleicacid complex in order to increase an amount of the single-strandednucleic acid.

In the method for producing a nucleic acid aptamer without targetimmobilization, selection of the target specific aptamer is carried outby, for example, repeatedly carrying out a graphene-based selectionprocess and a counter selection process one or more times using thetarget material and the counter-target material with respect to asingle-stranded nucleic acid specifically bound to the target material.

Herein, by the graphene-based counter selection process, it is possibleto remove a target non-specific single-stranded nucleic acid bound tothe counter target material and select a single-stranded nucleic acidwhich is not bound to the counter target material but bound to thegraphene.

Although not limited hereto, as a method for separating thesingle-stranded nucleic acid, which is not bound to a counter-targetmaterial but bound to graphene, from the graphene, a method forseparating a target specific aptamer from graphene by causing aconformational change by a main target with respect to a single-strandednucleic acid bound to graphene may be used.

Each step of the method for producing a nucleic acid aptamer withouttarget immobilization of the present invention will be explained indetail as follows with reference to FIG. 1.

In a first step, a single-stranded nucleic acid pool including a PCRprimer region at both ends and any 30 to 50 bases at its center is mixedwith a target material or a counter-target material in a buffersolution, and these are induced to be bound to each other at normaltemperature.

To be more specific, a nucleic acid pool of approximately 66 mersincluding binding sites (about 18 mers each) of a primer for PCRamplification at both ends and about 30 bases at its center issynthesized, and the nucleic acid pool is mixed with a target materialor a counter-target material in a buffer solution to make a bindingreaction.

The single-stranded nucleic acid pool may include, but is notparticularly limited to, a primer region set forth in SEQ ID NO: 1 atits 5′ end and a primer region set forth in SEQ ID NO: 2 at its 3′ end.

The target material may include, but is not particularly limited to, aprotein such as a Nampt protein having a molecular weight of 5000 to500000 daltons, a low molecular material having a molecular weight of 10to 2000 daltons, or a virus such as Bovine Viral Diarrhea Virus type 1.

The counter-target material may be a substance structurally andfunctionally similar to the target material and may be appropriatelyselected and used depending on a kind of the target material withoutparticular limitation. For example, if the target material is a Namptprotein, similar substances such as adiponectin, RBP4 (Retinol BindingProtein 4), resistin, vaspin, and HSA (Human Serum Albumin) may be used.If the target material is a Bovine Viral Diarrhea Virus type 1, similarsubstances such as Bovine Viral Diarrhea Virus type 2 (BVDV type 2),CSFV (Classical Swine Fever Virus), MDBK (Mardin-Darby Bovine Kidneycell), or BSA (Bovine Serum Albumin) may be used.

In a second step, the mixture is reacted with graphene to remove asingle-stranded nucleic acid which is not bound to the target materialor the counter-target material.

If graphene is added to a mixture of the single-stranded nucleic acidpool and the target material, a target non-specific single-strandednucleic acid which is not bound to the target material is bound to thegraphene and thus removed by centrifugation, and a supernatant includinga target material/single-stranded nucleic acid complex is taken toobtain a nucleic acid bound to the target by an ethanol precipitationmethod.

If graphene is added to a mixture of the single-stranded nucleic acidpool and the counter-target material, a target specific single-strandednucleic acid which is not bound to the counter-target material is boundto the graphene. Therefore, a target specific nucleic acid aptamer isseparated from the graphene by causing a conformational change by thetarget with respect to the target specific single-stranded nucleic acidbound to the graphene.

In a third step, a single-stranded nucleic acid specifically bound tothe target material and obtained in the above step is amplified byperforming a PCR using the PCR primer region.

A PCR is performed to amplify the single-stranded nucleic acidspecifically bound to the target material, a PCR product is purified,and then electrophoresis is carried out with polyacrylamide gelcontaining a high concentration of urea to separate the PCR product as adouble-stranded nucleic acid into two single-stranded nucleic acids. Inthis case, since the PCR product is a double-stranded nucleic acid, alabel is immobilized to a primer in order to perform a denaturationprocess for separating the double-stranded nucleic acid intosingle-stranded nucleic acids. During the electrophoresis, thedouble-stranded nucleic acid is denatured, and a nucleic acid strandwith the label and a nucleic acid strand without the label arepositioned up and down, respectively. The nucleic acid band with thelabel is cut off and extracted from the polyacrylamide gel, and then theseparated nucleic acid is obtained by performing the ethanolprecipitation method again.

A primer pair for the PCR may be set forth in SEQ ID NOS: 1 and 3, butis not particularly limited thereto.

The label may be a fluorescent material such as, but not particularlylimited to, Cy5, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 658,Cyanine-3, Cyanine-5, fluorescein, bodipy, Texas red, FITC (FluoresceinIsothiocyanate), rhodamine, or the like.

In a fourth step, a graphene-based selection process and a counterselection process are carried out repeatedly one or more times on thesingle-stranded nucleic acid specifically bound to the target materialusing the target material and the counter-target material.

The single-stranded nucleic acid pool specifically bound to the targetmaterial and obtained in the above step is reacted with the targetmaterial to increase affinity and a binding property with respect to thetarget. The selection process including a series of processes is carriedout several times. Then, the counter selection process is carried out byusing the counter-target material to block a nucleic acid which is notspecifically bound to the counter-target material. Thus, a nucleic acidpool specifically bound to the target material only with high affinityis obtained through the selection process and the counter selectionprocess.

In the counter selection process, if graphene is added after the countertarget material is reacted with the nucleic acid pool, a single-strandednucleic acid which is not bound to the counter-target material isadsorbed onto a surface of the graphene.

In a fifth step, a target non-specific single-stranded nucleic acidbound to the counter-target material in the graphene-based selectionprocess is removed, and a target specific aptamer is separated from thegraphene by causing a conformational change by the target materialthrough a reaction between a target specific single-stranded nucleicacid bound to the graphene and the target.

As for a single-stranded nucleic acid which is not bound to thecounter-target material, a supernatant is removed by centrifugation andonly graphene is taken out to obtain a single-stranded nucleic acidadsorbed onto the graphene from the graphene. To do so, the targetmaterial is reacted on the surface of the graphene onto which thesingle-stranded nucleic acid is adsorbed, and the target material causesa conformational change of the single-stranded nucleic acid adsorbedonto the graphene to effectively separate the single-stranded nucleicacid from the surface of the graphene. The single-stranded nucleic acidseparated as such becomes a candidate for an aptamer which is not boundto a counter target but strongly bound to a main target material.

The candidate for an aptamer is cloned using a cloning vector such as apDrive Cloning Vector (Qiagen, Netherlands), and a nucleic acid isextracted from a resultant colony to carry out a base sequence analysis.

In a method for comparing specific binding capacities of an aptamerspecifically bound to a target with respect to other proteins andmagnitudes of binding force depending on a concentration of a targetmaterial, a surface magnetic resonance device (SPR) may be used. To doso, a surface of a bare gold chip is modified and each DNA aptamer isimmobilized onto the chip. Then, a target material is reacted thereon atvarious concentrations and binding force thereof is checked and alsobinding force with respect to other counter-target materials is testedfor analyzing specific binding capacities.

A nucleic acid aptamer having a high target binding force is selected tobe used for detecting and separating the target.

A nucleic acid aptamer selected by the method for producing nucleic acidaptamer without target immobilization of the present invention mayinclude a nucleic acid aptamer having a sequence selected from the groupconsisting of the base sequences set forth in SEQ ID NOS: 4 to 28 inwhich the nucleic acid aptamer can be specifically bound to a Namptprotein or a Bovine Viral Diarrhea Virus type 1, but is not particularlylimited thereto since it may vary depending on a kind of a target.

According to another aspect of the present invention, the presentinvention provides a specific aptamer selected by the method forproducing a nucleic acid aptamer without target immobilization and a usethereof.

Therefore, the present invention relates to a nucleic acid aptamerhaving a sequence selected from the group consisting of the basesequences set forth in SEQ ID NOS: 4 to 18 in which the nucleic acidaptamer is specifically bound to a Nampt protein.

The aptamer of the present invention is selected to target a Namptprotein, and the Nampt protein may be that of a mammal such as a humanor a mouse, and more preferably, may be extracellular human Namptprotein (visfatin/PBEF) and may include amino acid sequences of 1 to491.

Further, the Nampt protein used as a target may include a peptidesynthesized by a publicly known method for synthesizing peptides, acommercially available Nampt protein such as a Nampt protein (Prod. No.AG-40A-0031) of AdipoGen Inc., and a whole or a part of the Namptprotein expressed from a recombinant vector manufactured by introducinga coding gene of the protein into a vector (for example, pAGNF, etc.),but is not particularly limited thereto.

Further, the expression “specifically bound to a Nampt protein” meansbeing specifically bound to the Nampt protein defined above by acovalent bond or a noncovalent bond.

The nucleic acid aptamer may be a DNA aptamer which is selected by theGO-SELEX method and specifically bound to a Nampt protein and has acertain base sequence, and preferably, a sequence selected from thegroup consisting of the base sequences set forth in SEQ ID NOS: 4 to 18.

The aptamer of the present invention can be chemically synthesized by amethod publicly known in the art.

The aptamer of the present invention may include a modified glycosylunit (for example, ribose or dioxyribose) of each nucleotide to increasea binding capacity with respect to a Nampt protein, stability, etc. At amodified site in the glycosyl unit, an oxygen atom at, for example, a 2′site, a 3′ site, and/or a 4′ site in the glycosyl unit may besubstituted with other atoms. The modification may include, for example,fluorination, O-alkylation (for example, O-methylation andO-ethylation), O-allylation, S-alkylation (for example, S-methylationand S-ethylation), S-allylation, and amination (for example, —NH). Suchmodification of the glycosyl unit is carried out by a publicly knownmethod (for example, refer to Sproat et al., (1991) Nucle. Acid. Res.19, 733-738; Cotton et al., (1991) Nucl. Acid. Res. 19, 2629-2635; Hobbset al., (1973) Biochemistry 12, 5138-5145).

The aptamer of the present invention may include a modified (forexample, chemically substituted) nucleic acid base (for example, purineand pyrimidine) to increase a binding capacity with respect to a Namptprotein, and the like. The modification may include, for example,5-position pyrimidine modification, 6- and/or 8-position purinemodification, modification at an exocyclic amine, substitution with4-thiouridine, and substitution with 5-bromo- or 5-iodo-uricyl.

Further, a phosphate group contained in the aptamer of the presentinvention may be modified to be resistant to nuclease and hydrolysis.For example, the P(0)0 group may be substituted with P(0)S (thioate),P(S)S (dithioate), P(0)NR₂ (amidate), P(0)R, P(0)OR′, CO or CH₂(formacetal) or 3′-amine (—NH—CH₂—CH₂—). Herein, each R or R′ isindependently H or a substituted or unsubstituted alkyl (for example,methyl and ethyl). Linkage groups may be —O—, —N—, or —S— linkage andcan be bonded to adjacent nucleotides through the —O—, —N—, or —S—linkage.

The modification of the present invention may further include 3′ and 5′modifications such as capping.

Further, the modification may be carried out by adding polyethyleneglycol, an amino acid, a peptide, inverted dT, a nucleic acid, anucleoside, myristoyl, lithocolic-oleyl, docosanyl, lauroyl, stearoyl,palmitoyl, oleoyl, linoleoyl, other lipids, a steroid, cholesterol,caffeine, a vitamin, a pigment, a fluorescent material, an anti-cancerdrug, a toxin, an enzyme, a radioactive material, biotin, etc. to anend. Regarding such modifications, refer to, for example, U.S. Pat. No.5,660,985 and U.S. Pat. No. 5,756,703.

The present invention further relates to a composition for detecting aNampt protein comprising: a nucleic acid aptamer having a sequenceselected from the group consisting of the base sequences set forth inSEQ ID NOS: 4 to 18 in which the nucleic acid aptamer is specificallybound to a Nampt protein.

Furthermore, the present invention relates to a method for detecting aNampt protein comprising: bringing the nucleic acid aptamer in contactwith a Nampt protein-containing sample to detect the Nampt protein.

Using a formation of a complex of the aptamer of the present inventionand a Nampt protein by a covalent bond or a noncovalent bond, it ispossible to detect the Nampt protein. The detection can be carried outby the same method as the immunological method except that the aptamerof the present invention is used instead of an antibody. Therefore,using the aptamer of the present invention instead of an antibody,detection and quantitation can be carried out by a method such as anenzyme immune-assay (EIA) (for example, competitive direct ELISA,competitive indirect ELISA, and sandwich ELISA), a radioimmuno assay(RIA), a fluorescence immuno assay (FIA), western blotting (for example,used as a secondary antibody in western blotting), animmunohistochemical staining method, a cell sorting method, etc.

The present invention further relates to a composition for diagnosing aNampt related disease comprising: a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 4 to 18 in which the nucleic acid aptamer isspecifically bound to a Nampt protein.

The Nampt related disease may include, for example, type 2 diabetes,colorectal cancer, prostate cancer, breast cancer, stomach cancer,polycystic ovarian syndrome, chronic renal failure, or chronicobstructive lung disease.

Diagnosis of the Nampt related disease may comprise bringing the aptamerin contact with a sample selected from tissue, cells, blood, serum,plasma, saliva, sputum, and urine. It is obvious that the sample is notlimited to the above-described examples as long as it is separated froma mammal, preferably a human, and contains Nampt as a biomarker such asa sample obtained by minimal invasion or a secretory body fluid sample,an in vitro cell culture medium sample, etc. More preferably, the samplemay be blood. For diagnosis of the Nampt related diseases, it is veryimportant to accurately measure a concentration of Nampt as an importantbiomarker for the Nampt related diseases in blood. Therefore, thecomposition containing the nucleic acid aptamer of the present inventionwhich is not specific to other various biomaterials in a blood samplebut can be specifically bound to Nampt even in a small amount can beused to diagnose the Nampt related diseases more rapidly and accurately.

If the aptamer is brought in contact with the sample, a specific bond isformed between Nampt as a biomarker present in the sample and theaptamer. Therefore, it is possible to detect a Nampt related disease bylabeling the aptamer with fluorescence and allowing the aptamer to bebound, and checking whether there is a signal or not.

The present invention further relates to a solid phase carrierimmobilizing a nucleic acid aptamer having a sequence selected from thegroup consisting of the base sequences set forth in SEQ ID NOS: 4 to 18in which the nucleic acid aptamer is specifically bound to a Namptprotein.

The solid phase carrier may include a substrate, a resin, a plate (forexample, a multiwall plate), a filter, a cartridge, a column, or aporous material.

The substrate may be employed from one used in a DNA chip or a proteinchip and may include, for example, a nickel-PTFE(polytetrafluoroethylene) substrate, a glass substrate, an apatitesubstrate, a silicon substrate, a gold substrate, a silver substrate, oran alumina substrate, and a polymer or the like may be coated on thesubstrate.

The resin may include agarose particles, silica particles, a copolymerof acrylamide and N,N′-methylenebisacrylamide, polystyrene-crosslinkeddivinylbenzene particles, epichlorohydrin-crosslinked dextran particles,a cellulose fiber, a crosslinked polymer of allyldextran andN,N′-methylenebisacrylamide, a monodispersed synthetic polymer, amonodispersed hydrophilic polymer, sepharose or Toyopearl, and the resinmay be bonded to various functional groups.

The solid phase carrier can be used to separate, purify, detect orquantitate the Nampt protein.

The aptamer of the present invention may be immobilized to a solid phasecarrier by a publicly known method. For example, a method in which anaffinity substance or a certain functional group is introduced into theaptamer of the present invention and the affinity substance or thecertain functional group is immobilized to a solid phase carrier may beused. The certain functional group may be a functional group which canbe provided in a coupling reaction, and may include, for example, anamino group, a thiol group, a hydroxyl group, and a carboxyl group. Forexample, biotin may be bound to an end of the aptamer to form a complex,and streptavidin may be immobilized to a surface of a substrate such asa chip, and thus the aptamer can be immobilized to the surface of thesubstrate by an interaction between the biotin and the streptavidinimmobilized to the surface of the substrate.

In a method for detecting a Nampt protein using a solid phase carrierimmobilizing an aptamer, for example, the nucleic acid aptamer of thepresent invention is spotted on a chip together with a fluorescentmaterial, a staining material, or an antibody and then reacts with ablood sample, whereby it is possible to rapidly measure a detectionsample, for example, a trace of Nampt contained in the blood. In anothermethod, if Nampt is bound to magnetic beads by immobilizing the nucleicacid aptamer to the magnetic beads, the bound nucleic acid aptamer/Namptcomplex can be separated using a magnet, and Nampt only can beselectively detected by again separating Nampt from the complex. Inaddition to the methods described in the exemplary embodiments of thepresent invention, a method in which Nampt in blood can be detectedusing a sensor linked with the nucleic acid aptamer of the presentinvention by a linkage may be used.

The detection of the Nampt protein using the solid phase carrier can beused to diagnose Nampt related diseases.

Further, the present invention can be provided in the form of a kit fordetecting a Nampt protein or a kit for diagnosing a Nampt relateddisease comprising: a nucleic acid aptamer having a sequence selectedfrom the group consisting of the base sequences set forth in SEQ ID NOS:4 to 18 in which the nucleic acid aptamer is specifically bound to aNampt protein.

The kit may include, if necessary, a buffer solution and containers fordetection and analysis in the form of bottles, tubs, sachets, envelopes,tubes, ampoules, etc. which may be partially or entirely made ofplastic, glass, paper, foil, wax, etc. The container may be equippedwith a stopper which may be a part of the container or can be attachedto the container mechanically or by means of adhesion or other means andcan be entirely or partially detached from the container. Further, thecontainer may be equipped with a stopper which can approach the contentsvia an injection needle. The kit may include an exterior package, andthe exterior package may include an instruction manual about use of thecomponents.

The aptamer specifically bound to a Nampt protein according to thepresent invention specifically detects the Nampt protein only.Therefore, it is obvious to one of ordinary skill in the art that acomposition for separating the Nampt protein comprising the same can beprovided.

Therefore, the present invention provides a composition for separating aNampt protein comprising: a nucleic acid aptamer having a sequenceselected from the group consisting of the base sequences set forth inSEQ ID NOS: 4 to 18 in which the nucleic acid aptamer is specificallybound to a Nampt protein.

Further, the present invention provides a Nampt protein separationmethod comprising: bringing a Nampt protein-containing sample in contactwith a solid phase carrier immobilizing a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 4 to 18 in which the nucleic acid aptamer isspecifically bound to a Nampt protein; and eluting the Nampt proteinbound to the solid phase carrier using an eluent.

The Nampt protein can be bound to the solid phase carrier of the presentinvention by a publicly known method. For example, a Namptprotein-containing sample (for example, bacteria, a culture or a culturesupernatant of cells, or blood) may be introduced to the solid phasecarrier of the present invention or another material containing thesame.

The elution of the Nampt protein can be carried out using an eluent suchas a neutral solution. A neutral eluent is not particularly limited andmay have, for example, a pH of about 6 to about 9, preferably about 6.5to about 8.5, and more preferably about 7 to about 8. The neutralsolution may contain potassium salts (for example, NaCl and KCl),magnesium salts (for example, MgCl), surfactants (for example, Tween 20,Triton, and NP40), or glycerin.

The separation method of the present invention may comprise a processfor cleaning the solid phase carrier with a cleaning solution after theNampt protein is bound. The cleaning solution may include urea, achelate agent (for example, EDTA), Tris, an acid, or an alkali.

The separation method of the present invention may comprise a processfor heating the solid phase carrier. Through this process, the solidphase carrier can be regenerated and sterilized.

According to another aspect of the present invention, the presentinvention relates to a nucleic acid aptamer having a sequence selectedfrom the group consisting of the base sequences set forth in SEQ ID NOS:19 to 28 in which the nucleic acid aptamer is specifically bound to aBovine Viral Diarrhea Virus type 1.

The aptamer of the present invention is selected by the GO-SELEX methodto target a Bovine Viral Diarrhea Virus type 1 and may be a DNA aptamerwhich is specifically bound to the virus and has a certain basesequence, and preferably a sequence selected from the group consistingof the base sequences set forth in SEQ ID NOS: 19 to 28.

The aptamer of the present invention can be chemically synthesized by amethod publicly known in the art.

The aptamer of the present invention may include a modified glycosylunit (for example, ribose or dioxyribose) of each nucleotide to increasea binding capacity with respect to a Bovine Viral Diarrhea Virus type 1,stability, etc. A modified site in the glycosyl unit, a kind ofmodification, and a modification method of the glycosyl unit are thesame as described above.

The aptamer of the present invention may include a modified (forexample, chemically substituted) nucleic acid base (for example, purineand pyrimidine) to increase a binding capacity with respect to a BovineViral Diarrhea Virus type 1, and the like. A kind of this modificationis the same as described above.

Further, a phosphate group contained in the aptamer of the presentinvention may be modified to be resistant to nuclease and hydrolysis. Akind of this modification is the same as described above.

The modification of the present invention may further include 3′ and 5′modifications such as capping. A kind of this modification is the sameas described above.

The present invention further relates to a composition for detecting aBovine Viral Diarrhea Virus type 1 comprising: a nucleic acid aptamerhaving a sequence selected from the group consisting of the basesequences set forth in SEQ ID NOS: 19 to 28 in which the nucleic acidaptamer is specifically bound to a Bovine Viral Diarrhea Virus type 1.

Furthermore, the present invention relates to a method for detecting aBovine Viral Diarrhea Virus type 1 comprising: bringing the nucleic acidaptamer having a sequence selected from the group consisting of the basesequences set forth in SEQ ID NOS: 19 to 28 in which the nucleic acidaptamer is specifically bound to a Bovine Viral Diarrhea Virus type 1 incontact with a Bovine Viral Diarrhea Virus type 1-containing sample todetect the Bovine Viral Diarrhea Virus type 1.

The Bovine Viral Diarrhea Virus type 1 may be detected from a samplecollected from any one of serum, blood, urine, water, tears, sweat,saliva, lymph, cerebrospinal fluid, soil, air, food, waste, animalintestines, and animal tissues, but is not limited thereto. Herein, thewater may include river water, seawater, lake water, and rain water. Thewaste may include sewage, wastewater, and the like. The animal mayinclude a cow. Further, the animal tissues may include mucous membranes,skin, cortices, hair, scales, eyes, tongue, cheeks, hooves, beaks,snouts, feet, hands, mouths, nipples, ears, noses, etc.

According to an exemplary embodiment of the present invention, as aresult of comparing binding capacity of an aptamer specific to a BovineViral Diarrhea Virus type 1 (BVDV type 1) and a magnitude of bindingforce depending on a concentration of the BVDV type 1 using a surfacemagnetic resonance device (SPR), the aptamer was found to exhibit a veryhigh binding force with respect to the BVDV type 1 (BVDV t1) but not tobind well to other similar substances (CSFV, MDBK, and BSA) includingBVDV type 2 (BVDV t2). Such a result means that the nucleic acid aptamerspecific to the BVDV type 1 of the present invention can specificallydetect the BVDV type 1 and also enables bovine viral diarrhea to bediagnosed more accurately.

Therefore, the present invention relates to a composition for diagnosingbovine viral diarrhea comprising: a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 19 to 28 in which the nucleic acid aptamer isspecifically bound to a Bovine Viral Diarrhea Virus type 1.

The diagnosis of bovine viral diarrhea is carried out to diagnose bovineviral diarrhea of animals other than humans, particularly cows.

Further, the nucleic acid aptamer specific to a BVDV type 1 of thepresent invention can be used to detect the BVDV type 1 or to diagnosebovine viral diarrhea by a sandwich binding method using a first aptamerand a second aptamer. For example, it is possible to detect a BVDV type1 or diagnose bovine viral diarrhea by bringing a Bovine Viral DiarrheaVirus type 1-containing sample in contact with a solid phase carrierimmobilizing a first nucleic acid aptamer specific to the Bovine ViralDiarrhea Virus type 1, adding a second nucleic acid aptamer specific tothe Bovine Viral Diarrhea Virus type 1 to detect whether or not asandwich complex of the first nucleic acid aptamer, the Bovine ViralDiarrhea Virus type 1, and the second nucleic acid aptamer is formed.

The first nucleic acid aptamer is immobilized to the solid phase carrierand used to be bound to the Bovine Viral Diarrhea Virus type1-containing sample and may have a base sequence set forth in SEQ IDNOS: 23, but any aptamer specific to the Bovine Viral Diarrhea Virustype 1 can be used without limitation.

The second nucleic acid aptamer can be used to be bound to a complex ofthe first nucleic acid aptamer and the Bovine Viral Diarrhea Virus type1, and since it is bound to a label selected from the group consistingof a fluorescent material, a quantum dot, a radioactive label, a goldnanoparticle, an enzyme, an enzyme-substrate, and a electrochemicalfunctional group, it is possible to detect whether or not the sandwichcomplex of the first nucleic acid aptamer, the Bovine Viral DiarrheaVirus type 1, and the second nucleic acid aptamer is formed by detectingthe label or a reaction of the label so as to detect the BVDV type 1 ordiagnose bovine viral diarrhea.

The second nucleic acid aptamer may have a base sequence set forth inSEQ ID NOS: 26, but any aptamer specific to the Bovine Viral DiarrheaVirus type 1 can be used without limitation.

Any signal processing method may be used to check whether or not thesandwich complex of the first nucleic acid aptamer, the Bovine ViralDiarrhea Virus type 1, and the second nucleic acid aptamer is formed aslong as it can check a complex, and it can be appropriately useddepending on, for example, a kind of a label.

The detection of the label or a reaction of the label may be carried outby a generally known method for analyzing a label or a reaction thereof.For example, if a fluorescent material is used as a label, luminescenceor color change occurs when a target material is present. Therefore, bymeasuring this, the target material can be detected. For example, it ispossible to check whether the target material is detected or not byscanning a well in which a reaction occurs with an image scanner capableof detecting fluorescent pigments, and it is possible to measure adetectable amount by measuring a concentration from the image withsoftware.

The present invention further relates to a solid phase carrierimmobilizing a nucleic acid aptamer having a sequence selected from thegroup consisting of the base sequences set forth in SEQ ID NOS: 19 to 28in which the nucleic acid aptamer is specifically bound to a BovineViral Diarrhea Virus type 1.

A kind of the solid phase carrier and a method for immobilizing theaptamer are the same as described above.

The solid phase carrier of the invention can be used to purify,separate, detect or quantitate the Bovine Viral Diarrhea Virus type 1.

A method for detecting a Bovine Viral Diarrhea Virus type 1 using thesolid phase carrier immobilizing the aptamer is the same as describedabove.

The detection of the Bovine Viral Diarrhea Virus type 1 using the solidphase carrier can be used to diagnose bovine viral diarrhea.

Further, the present invention can be provided in the form of a kit fordetecting a Bovine Viral Diarrhea Virus type 1 or a kit for diagnosingbovine viral diarrhea comprising: a nucleic acid aptamer which has anyone of base sequences of SEQ ID NOS: 19 to 28 and can be specificallybound to the Bovine Viral Diarrhea Virus type 1.

The kit for detecting a Bovine Viral Diarrhea Virus type 1 or fordiagnosing bovine viral diarrhea may be provided in the form of bottles,tubs, sachets, envelopes, tubes, ampoules, etc. which may be partiallyor entirely made of plastic, glass, paper, foil, wax, etc. Such acontainer may be equipped with a stopper which may be a part of thecontainer or can be attached to the container mechanically or by meansof adhesion or other means and can be entirely or partially detachedfrom the container. Further, the container may be equipped with astopper which can approach the contents via an injection needle. The kitmay include an exterior package, and the exterior package may include aninstruction manual about use of the components.

The aptamer specifically bound to the Bovine Viral Diarrhea Virus type 1according to the present invention specifically detects the Bovine ViralDiarrhea Virus type 1 only. Therefore, it is obvious to one of ordinaryskill in the art that a composition for separating the Bovine ViralDiarrhea Virus type 1 comprising the same can be provided.

Therefore, the present invention relates to a composition for separatinga Bovine Viral Diarrhea Virus type 1 comprising: a nucleic acid aptamerhaving a sequence selected from the group consisting of the basesequences set forth in SEQ ID NOS: 19 to 28 in which the nucleic acidaptamer is specifically bound to a Bovine Viral Diarrhea Virus type 1.

Further, the present invention provides a Bovine Viral Diarrhea Virustype 1 separation method comprising: bringing a Bovine Viral DiarrheaVirus type 1-containing sample in contact with a solid phase carrierimmobilizing a nucleic acid aptamer having a sequence selected from thegroup consisting of the base sequences set forth in SEQ ID NOS: 19 to 28in which the nucleic acid aptamer is specifically bound to a BovineViral Diarrhea Virus type 1; and eluting the Bovine Viral Diarrhea Virustype 1 bound to the solid phase carrier using an eluent.

According to an aspect of the present invention, preferably, a column isfilled with beads immobilizing the aptamer and a Bovine Viral DiarrheaVirus type 1-containing sample passes through the column, whereby it ispossible to separate the Bovine Viral Diarrhea Virus type 1.

Hereinafter, the present invention will be described in detail by meansof Examples. However, it should be understood that the following Exampleare given by way of illustration of the present invention only, and arenot intended to limit the scope of the present invention.

Example 1 Selection of Nampt Specific Aptamer

As a DNA pool of 66 mers, a DNA pool including a PCR primer region atboth ends and any 30 bases at its center was synthesized.

5′-CGTACGGAATTCGCTAGC-N30-GGATCCGAGCTCCACGTG-3′(SEQ ID NO: 1: CGTACGGAATTCGCTAGC; SEQ ID NO: 2: GGATCCGAGCTCCACGTG)

This DNA pool was put into a buffer solution (pH 7.4, 20 mM Tris, 100 mMNaCl, 2 mM MgCl₂) and mixed with Nampt (a Nampt protein produced byAdipoGen Inc., Prod. No. AG-40A-0031) in the same amount and reacted atnormal temperature for 30 minutes. Then, in order to remove DNA whichwas not bound to Nampt, the mixture solution was reacted with a graphenesolution for 2 hours. In this case, a single-stranded DNA which was notbound to a target was strongly adsorbed onto a surface of graphene byπ-stacking.

The graphene was separated through centrifugation, and a supernatant wastaken out to obtain a single-stranded DNA bound to the target by anethanol precipitation method. An amount of the DNA bound to Namptobtained as such was measured using a spectrophotometer.

In order to increase the amount of the DNA specifically bound to Nampt,a PCR was carried out using the already-known primer regions. The PCRproduct is a double-stranded DNA, and thus fluorescein was immobilizedto a primer in order to separate the double-stranded DNA intosingle-stranded DNAs.

Forward primer: (SEQ ID NO: 1) 5′-fluorescein-CGTACGGAATTCGCTAGC-3′Reverse primer: (SEQ ID NO: 3) 5′-CACGTGGAGCTCGGATCC-3′

After the PCR product was purified using a purification kit,polyacrylamide gel electrophoresis was carried out to separate thedouble-stranded DNA into single-stranded DNAs. 10% polyacrylamide gelcontained 6 M urea and 20% formamide, and thus, after theelectrophoresis, two bands were formed. During the electrophoresis, thedouble-stranded DNA was denatured, and a DNA strand with fluorescein anda DNA strand without the fluorescein were positioned up and down,respectively.

The DNA band with fluorescein was cut off and extracted from thepolyacrylamide gel, and then the separated DNA was obtained byperforming the ethanol precipitation method again. The DNA pool obtainedas such was reacted with Nampt again. A schematic diagram of such aprocess is shown in FIG. 1. A selection process including a series ofprocesses was carried out five times in total to develop an aptamer.After the fourth selection process, a counter selection process wascarried out with HSA and other adipokines such as adiponectin, RBP4(Retinol Binding Protein 4), resistin, and vaspin to block a DNA whichwas not specifically bound and obtain a DNA pool which was specificallybound to Nampt only with high affinity. In the counter selectionprocess, unlike a general process, instead of Nampt, the above-describedcounter targets were reacted with the DNA pool in the same buffer for 30minutes and then reacted with a graphene solution for 2 hours. In thisprocess, a single-stranded DNA which was not bound to a counter targetwas adsorbed onto a surface of graphene. A supernatant was removed bycentrifugation and only graphene was taken out to obtain asingle-stranded DNA adsorbed onto the graphene from the graphene. To doso, the Nampt was reacted on the surface of the graphene onto which thesingle-stranded DNA was adsorbed, and the Nampt as a main target causeda conformational change of the single-stranded DNA adsorbed onto thegraphene to effectively separate the single-stranded DNA from thesurface of the graphene. The single-stranded DNA separated as suchbecame a candidate for an aptamer which was not bound to the countertarget but strongly bound to the Nampt as a main target.

FIG. 2 illustrates a percentage of single-stranded DNA bound to Nampt ineach selection round.

The DNA pool finally obtained was cloned using a pDrive Cloning Vector,and DNA was extracted from a resultant colony to carry out a basesequence analysis. As a result, 15 kinds of DNAs specifically bound toNampt were obtained.

A result of analysis on base sequences of the 15 kinds of DNAsspecifically bound to Nampt with high affinity is shown in Table 1.Further, a result of prediction about secondary structures of these 15kinds of aptamers using the M-fold program is shown in FIGS. 3 to 17.

TABLE 1 SEQ ID Aptamer NOS No. Sequence (5′-3′)  4 G56CGTACGGAATTCGCTAGCCCGTGGGGTAGCGGG GTCGTGTGATATGTGGATCCGAGCTCCACGTG  5G12 CGTACGGAATTCGCTAGCGGGTGCCGTGGCACG AGGCCGTGGTCCAGGGGATCCGAGCTCCACGTG 6 G4  CGTACGGAATTCGCTAGCGTGATGTGGGGGTACGCTCGTGGCAGGCTTGGATCCGAGCTCCACGTG  7 G27CGTACGGAATTCGCTAGCGGGTGGAGTACGTGG GGGTCATCCTGTGTGGGATCCGAGCTCCACGTG  8G35 CGTACGGAATTCGCTAGCGGTGACGGACGTGGG GTGCACGAAGGGAGGGGATCCGAGCTCCACGTG 9 G26 CGTACGGAATTCGCTAGCATCGGGTGCAGAGTCGGAGCTAACGGCAGCGGATCCGAGCTCCACGTG 10 G9 CGTACGGAATTCGCTAGCGGGGATGGGCCGCTC TGCAGAATGTTCTGTGGATCCGAGCTCCACGTG 11G32 CGTACGGAATTCGCTAGCGTGGACTGGCGGAAA TCTTGGTATGCCCATGGATCCGAGCTCCACGTG12 G21 CGTACGGAATTCGCTAGCGGGTTCGGGACGGATGAACGTGATAGCTGAGGATCCGAGCTCCACGTG 13 G15CGTACGGAATTCGCTAGCAGCCGGCGGGTGCTC AATGTTGGGGGTTGGGATCCGAGCTCCACGTG 14G40 CGTACGGAATTCGCTAGCCGGGGTGGGAACCAG TCTTGCGCGGGTGACGGATCCGAGCTCCACGTG15 G54 CGTACGGAATTCGCTAGCGTGGCGGGGCGCGGGTGCCGGAGTTGATGTGGATCCGAGCTCCACGTG 16 G37CGTACGGAATTCGCTAGCGGGCGATGTGCGGAA TGTGGGATTGCGGGTGGATCCGAGCTCCACGTG 17G39 CGTACGGAATTCGCTAGCGGTTGCCGTGCGGCG TGCGAGTTGGGCCTTGGATCCGAGCTCCACGTG18 G55 CGTACGGAATTCGCTAGCGGACTGGAGTCTAGACCGGGTAGCTGTGGTGGATCCGAGCTCCACGTG

Example 2 Analysis on Nampt Binding Specificity

An experiment showing that 15 kinds of aptamers strongly bound to Namptwere not bound to other blood proteins but specifically bound to Namptonly was carried out by immobilizing aptamers onto a gold chip with asurface magnetic resonance device (SPR). Since Nampt was one ofadipokines, as the most suitable controls to show that a Nampt aptamerwas specifically bound to Nampt only, other adipokines (RBP4,adiponectin, resistin, and vaspin) and HSA were used.

Firstly, a carboxyl group (—COOH) was formed on a surface of a gold chipwith 50 mM of 3,3′-dithiodipropionic acid, and a self-assembly monolayerwas formed with EDC/NHS. Then, streptavidin was immobilized thereon, andeach aptamer bound to biotin was immobilized. Herein, 200 nM Nampt andthe controls, i.e., RBP4, adiponectin, resistin, vaspin, and HAS, werereacted for 30 minutes in respective buffer solutions (100 mM NaCl, 2 mMMgCl₂, 5 mM KCl, 1 mM CaCl₂, and 20 mM Tris-Cl buffer solutioncontaining 0.02% Tween 20, pH 7.6), and non-bound proteins were washedoff with the same buffer solutions.

Finally, a result of the SPR was analyzed, and it could be confirmedthat the Nampt aptamers were superiorly bound to Nampt compared to otherproteins. This result is shown in FIG. 18.

Example 3 Measurement of Dissociation Constant (K_(d)) of Nampt

Aptamers G35, G40, and G55 had the highest specificity and binding forceamong the 15 kinds of aptamers specifically bound to Nampt and producedin Example 1. Binding of these aptamers with respect to Nampt wasanalyzed. The aptamers were immobilized onto a gold chip in the samemanner as described above, and Nampt at various concentrations of 20 nMto 400 nM was reacted for 30 minutes in buffer solutions (100 mM NaCl, 2mM MgCl₂, 5 mM KCl, 1 mM CaCl₂, and 20 mM Tris-Cl buffer solutioncontaining 0.02% Tween 20, pH 7.6). In order to obtain a dissociationconstant, each reaction level was plotted by a nonlinear regressionmethod and a single site saturation ligand binding method with GraphpadPrism 5.0. In this case, the equation Y=B_(max)*X̂h/(K_(d)̂h+X̂h) was used(y represents a saturation degree, B_(max) represents a maximal bindingsite, K_(d) represents a dissociation constant, X represents non-boundNampt, and h represents a hill slope constant).

As a result, the K_(d) values of the aptamers G35, G40, and G55 were117.4 nM, 114.4 nM, and 71.7 nM, respectively, and thus it was confirmedthat they were strongly bound to Nampt. Analysis data thereof is shownin FIG. 19.

Example 4 Selection of Bovine Viral Diarrhea Virus Type 1 SpecificAptamer

As a DNA pool of 66 mers, a DNA pool including a PCR primer region atboth ends and any 30 bases at its center was synthesized.

5′-CGTACGGAATTCGCTAGC-N30-GGATCCGAGCTCCACGTG-3′(SEQ ID NO: 1: CGTACGGAATTCGCTAGC; SEQ ID NO: 2: GGATCCGAGCTCCACGTG)(N: A, T, G or C)

Firstly, the DNA pool was put into a buffer solution (pH 7.4, 20 mMTris, 100 mM NaCl, 2 mM MgCl₂) and mixed with similar substances of aBovine Viral Diarrhea Virus type 1 as a target material, i.e., BVDV type2, CSFV (Classical Swine Fever Virus), MDBK (Mardin-Darby Bovine Kidneycell), or BSA (Bovine Serum Albumin), and reacted at normal temperaturefor 30 minutes. Then, in order to remove DNA which was bound to thesimilar substances and obtain only DNA which was not bound to thesimilar substances, the mixture solution was reacted with a graphenesolution for 2 hours. In this case, a single-stranded DNA which was notbound to the similar substances was strongly adsorbed onto a surface ofgraphene by π-stacking. The DNA which was not bound to the similarsubstances but adsorbed onto the graphene became a candidate for a BVDVtype 1 aptamer.

The graphene was separated through centrifugation, a supernatant wasdiscarded, and the separated graphene was washed off three times withthe same buffer solution. The BVDV type 1 was put into the washedgraphene as a target material and reacted therewith for 2 hours. In thiscase, the target material was reacted with the DNA adsorbed onto thesurface of the graphene and caused a conformational change of DNA, andthe conformationally changed DNA was separated from the graphene.

Then, a single-stranded DNA bound to the target was obtained from theDNA separated from the graphene by an ethanol precipitation method. Anamount of the DNA bound to the BVDV type 1 obtained as such was measuredusing a spectrophotometer.

In order to amplify the DNA specifically bound to the BVDV type 1, a PCRwas carried out using the already-known primer regions. The PCR productis a double-stranded DNA, and thus fluorescein was immobilized to aprimer in order to separate the double-stranded DNA into single-strandedDNAs.

Forward primer: (SEQ ID NO: 1) 5′FP-fluorescein-CGTACGGAATTCGCTAGC-3′Reverse primer: (SEQ ID NO: 3) 5′RP-CACGTGGAGCTCGGATCC-3′

After the PCR product was purified using a purification kit,polyacrylamide gel electrophoresis was carried out to separate thedouble-stranded DNA into single-stranded DNAs. 10% polyacrylamide gelcontained 6 M urea and 20% formamide, and thus, after theelectrophoresis, two bands were formed. During the electrophoresis, thedouble-stranded DNA was denatured, and a DNA strand with fluorescein anda DNA strand without the fluorescein were positioned up and down,respectively.

The DNA band with fluorescein was cut off to carry out gel extraction,and then the separated DNA was obtained by performing the ethanolprecipitation method again. The DNA pool obtained as such was reactedwith the similar substances of the BVDV type 1 again. Such a series ofprocesses was repeated. A selection process was carried out five timesin total to develop an aptamer. Since DNA which could be bound to thesimilar substances was removed in each selection process, the aptamerhad high selectivity. Further, since DNA was taken out by aconformational change caused by the target material in each process, itwas possible to obtain the aptamer having high affinity.

FIG. 20 illustrates a percentage of single-stranded DNA bound to a BVDVtype 1 as a target material in each selection round.

The DNA pool finally obtained was cloned using a pDrive Cloning Vector(Qiagen, Netherlands), and DNA was extracted from a resultant colony tocarry out a base sequence analysis. As a result, 10 kinds of DNAsspecially bound to the BVDV type 1 were obtained. Base sequences of theDNAs were analyzed, and a result thereof is shown in Table 2.

Further, a result of prediction about secondary structures of these 10kinds of aptamers using the M-fold program is shown in FIGS. 21 to 30.

TABLE 2 SEQ ID Aptamer NOS No. Sequence (5′-3′) 19 B1-13CGTACGGAATTCGCTAGCTTGGGTATAACGTTC TGAGTCAGAACGCCGGGATCCGAGCTCCACGTG 20B1-38 CGTACGGAATTCGCTAGCACGGCGGGTCGCCCGACTTTGGGCCGACTGGGATCCGAGCTCCACGTG 21 B1-34CGTACGGAATTCGCTAGCCGCTCGGGGCGCTGC ACGTAGGGTGGGGTGGGATCCGAGCTCCACGTG 22B1-17 CGTACGGAATTCGCTAGCGTGGGGCAGTTGCTTGATGATCTGTAGCGCGGATCCGAGCTCCACGTG 23 B1-11CGTACGGAATTCGCTAGCTGCGCATCCACAAAT GTATTGTCGGGGGATGGATCCGAGCTCCACGTG 24B1-39 CGTACGGAATTCGCTAGCCGGGTCTAGTCAGGAGGTTCCTGTGGTGCTGGATCCGAGCTCCACGTG 25 B1-8 CGTACGGAATTCGCTAGCGAGGGCGCCAAACAG GGTGTCCTGGCGTTGGGATCCGAGCTCCACGTG 26B1-43 CGTACGGAATTCGCTAGCTGCGGACTCGCGATGCTACTTCTGATGATAGGATCCGAGCTCCACGTG 27 B1-27CGTACGGAATTCGCTAGCTAAGGGTAGCACAGG TCACCTCGCCACTGTGGATCCGAGCTCCACGTG 28B1-40 CGTACGGAATTCGCTAGCCGAACGTTGCGGTGTGGAACTTCGCGAGCAGGATCCGAGCTCCACGTG

Example 5 Analysis on Bovine Viral Diarrhea Virus Binding Specificity

In order to confirm that aptamers specific to the 10 kinds of BovineViral Diarrhea Virus type 1 (BVDV type 1) separated in Example 4 did notshow specificity to other similar substances, i.e., BVDV type 2 that isthe most suitable control of the BVDV type 1, Classical Swine FeverVirus belonging to the same family, MDBK cells used to culture BVDV type1, and Bovine Serum Albumin, but acted specifically to the BVDV type 1,the following experiment was carried out.

In order to carry out an SPR (Surface Plasmon Resonance) analysis, —COOHwas formed on a surface of a gold chip with 50 mM 3,3′-dithiodipropionicacid, and then a self-assembly monolayer was formed with EDC/NHS. Then,streptavidin was immobilized thereon, and each aptamer of Example 4bound to biotin was immobilized. Herein, the BVDV type 1 in the sameamount and the similar substances (BVDV type 2, Classical Swine FeverVirus, MDBBK cells, and Bovine Serum Albumin) were reacted for 30minutes in buffer solutions (100 mM NaCl, 2 mM MgCl₂, 5 mM KCl, 1 mMCaCl₂, and 20 mM Tris-Cl buffer solution containing 0.02% Tween 20, pH7.6), respectively, and non-bound substances were washed off with thesame buffer solutions.

Thereafter, a result of the SPR was analyzed with an Autolab springle(single channel, Eco Chemie, Netherlands) and it could be confirmed thatall the 10 kinds of the aptamers showed high specificity to the BVDVtype 1, and affinity of 3 kinds of aptamers observed to have the highestspecificity to the BVDV type 1, i.e. an aptamer B1-11 (SEQ ID NO: 23),an aptamer B1-34 (SEQ ID NO: 21), and an aptamer B1-43 (SEQ ID NO: 26),is shown in FIG. 31.

As shown in FIG. 31, all of the aptamer B1-11 (SEQ ID NO: 23), theaptamer B1-34 (SEQ ID NO: 21), and the aptamer B1-43 (SEQ ID NO: 26)exhibited very high binding force with respect to the BVDV type 1 (BVDVt1) but did not bind well to the similar substances (CSFV, MDBK, andBSA) including BVDV type 2 (BVDV t2).

Such a result means that the nucleic acid aptamer specific to the BVDVtype 1 of the present invention can specifically detect the BVDV type 1and also makes it possible to diagnose bovine viral diarrhea moreaccurately.

Example 6 Measurement of Dissociation Constant (K_(d)) of Bovine ViralDiarrhea Virus Type 1

Aptamers B1-11, B1-34, and B1-43 had the highest specificity among the10 kinds of aptamers specifically bound to the BVDV type 1 and producedin Example 4. Binding of these aptamers with respect to the BVDV type 1was analyzed.

The aptamers were immobilized in the same manner as Example 3, and theBVDV type 1 at various concentrations was reacted for 30 minutes inbuffer solutions (100 mM NaCl, 2 mM MgCl₂, 5 mM KCl, 1 mM CaCl₂, and 20mM Tris-Cl buffer solution containing 0.02% Tween 20, pH 7.6). In orderto obtain a dissociation constant, each reaction level was plotted by anonlinear regression method and a single site saturation ligand bindingmethod with Graphpad Prism 5.0. In this case, the equationY=B_(max)*X̂h/(K_(d)̂h+X̂h) was used (y represents a saturation degree,B_(max) represents a maximal binding site, K_(d) represents adissociation constant, X represents non-bound Nampt, and h represents ahill slope constant).

As a result, the K_(d) values of the three aptamers were obtained, andanalysis data thereof is shown in FIG. 32.

FIG. 32 shows that all of the three aptamers exhibited high affinitywith respect to the BVDV type 1.

Example 7 Detection of Bovine Viral Diarrhea Virus by Sandwich BindingMethod

In order to find an optimum composition, i.e., a first aptamer (aptamerimmobilized on a solid phase) and a second aptamer (aptamer bound to atarget material bound to the first aptamer and labeled with a label), todetect a Bovine Viral Diarrhea Virus type 1 by a sandwich binding method(FIG. 33), the following experiment was carried out.

Firstly, with the three aptamers confirmed as having the highestaffinity in Examples 5 and 6, one of the three aptamers (the aptamerB1-11 (SEQ ID NO: 23), the aptamer B1-34 (SEQ ID NO: 21), and theaptamer B1-43 (SEQ ID NO: 26)) was immobilized onto a gold chip in thesame manner as Example 5 and bound to the BVDV type 1 and thenadditionally bound to any one of the three aptamers. Sequences of 9combinations in total were tested, and results thereof were compared.

As a result, when the aptamer B1-11 (SEQ ID NO: 23) having the lowestK_(d) was immobilized and the aptamer B1-43 (SEQ ID NO: 26) having thehighest K_(d) was reacted in a second-order reaction, the best resultcould be obtained as shown in FIG. 34. That is, in the case of asandwich binding in which the aptamer B1-11 with SEQ ID NO: 23 wasimmobilized as a first aptamer on a solid phase, and the BVDV type 1 asa target material was added thereto and reacted with the aptamer B1-43with SEQ ID NO: 26 as a second aptamer in a second-order reaction, theBVDV type 1 could be detected most sensitively.

The specific parts of the present invention have been described indetail. It is obvious to one of ordinary skill in the art that suchdetailed descriptions are merely provided to illustrate preferableembodiments and do not limit the scope of the present invention.

Therefore, the effective scope of the present invention shall be definedby the accompanying claims and their equivalents.

The present invention provides a technique capable of developing anaptamer using graphene without immobilizing a main target. Since thetarget is not immobilized, a binding site of the target is not limited,and thus it is possible to develop an aptamer having a high bindingforce with respect to the target. Further, since expensive andcomplicated equipment and skilled manpower are not needed, it ispossible to readily develop an aptamer.

Furthermore, a Nampt protein used as a target model can be effectivelydetected using an aptamer, and thus it is possible to measure aconcentration of the Nampt protein in blood more stably and sensitivelyas compared with an antibody-based analysis which has beenconventionally used. Moreover, a DNA aptamer requires less productioncosts than an antibody and is easily immobilized on a surface, and thusit is useful in manufacturing a biosensor chip. With a biosensor capableof sensitively and accurately measuring a concentration of a Namptprotein in blood using a DNA aptamer specifically bound to Nampt, it ispossible to more accurately diagnose Nampt related diseases such as type2 diabetes, colorectal cancer, prostate cancer, breast cancer, stomachcancer, polycystic ovarian syndrome, chronic renal failure, chronicobstructive lung disease, etc.

Also, since a biosensor capable of diagnosing bovine viral diarrhea isdeveloped using an aptamer specifically bound to a Bovine Viral DiarrheaVirus type 1 used as a target model, a more accurate diagnosis can beexpected.

In addition, a sandwich detection technique using two or more kinds ofthe aptamer to detect a Bovine Viral Diarrhea Virus type 1 is capable ofmore sensitively detecting the virus through signal enhancement, andthus it is possible to accurately diagnose bovine viral diarrhea.

The present invention can be used as a kit for detecting or diagnosingproteins, low molecular materials, viruses, or diseases related thereto.

While the invention has been shown and described with reference tocertain exemplary embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the spirit and scope of the invention asdefined by the appended claims.

What is claimed is:
 1. A nucleic acid aptamer having a sequence selectedfrom the group consisting of the base sequences set forth in SEQ ID NOS:19 to 28, wherein the nucleic acid aptamer is specifically bound to aBovine Viral Diarrhea Virus type
 1. 2. A composition for detecting aBovine Viral Diarrhea Virus type 1 comprising: a nucleic acid aptamerhaving a sequence selected from the group consisting of the basesequences set forth in SEQ ID NOS: 19 to 28, wherein the nucleic acidaptamer is specifically bound to a Bovine Viral Diarrhea Virus type 1.3. A Bovine Viral Diarrhea Virus type 1 detection method comprising:bringing a nucleic acid aptamer having a sequence selected from thegroup consisting of the base sequences set forth in SEQ ID NOS: 19 to 28in which the nucleic acid aptamer is specifically bound to a BovineViral Diarrhea Virus type 1 in contact with a Bovine Viral DiarrheaVirus type 1-containing sample to detect the Bovine Viral Diarrhea Virustype
 1. 4. The Bovine Viral Diarrhea Virus type 1 detection method ofclaim 3, wherein the sample is any one of serum, blood, urine, water,tears, sweat, saliva, lymph, cerebrospinal fluid, soil, air, food,waste, animal intestines, and animal tissues.
 5. The Bovine ViralDiarrhea Virus type 1 detection method of claim 3, wherein a BovineViral Diarrhea Virus type 1-containing sample is brought in contact witha solid phase carrier immobilizing a first nucleic acid aptamer specificto the Bovine Viral Diarrhea Virus type 1, and a second nucleic acidaptamer specific to the Bovine Viral Diarrhea Virus type 1 is added todetect whether or not a sandwich complex of the first nucleic acidaptamer, the Bovine Viral Diarrhea Virus type 1, and the second nucleicacid aptamer is formed.
 6. The Bovine Viral Diarrhea Virus type 1detection method of claim 5, wherein the first nucleic acid aptamer hasa base sequence set forth in SEQ ID NO: 23, and the second nucleic acidaptamer has a base sequence set forth in SEQ ID NO:
 26. 7. The BovineViral Diarrhea Virus type 1 detection method of claim 5, wherein thesecond nucleic acid aptamer is bound to a label selected from the groupconsisting of a fluorescent material, a quantum dot, a radioactivelabel, a gold nanoparticle, an enzyme, an enzyme-substrate, and aelectrochemical functional group.
 8. A composition for diagnosing bovineviral diarrhea comprising: a nucleic acid aptamer having a sequenceselected from the group consisting of the base sequences set forth inSEQ ID NOS: 19 to 28, wherein the nucleic acid aptamer is specificallybound to a Bovine Viral Diarrhea Virus type
 1. 9. A solid phase carrierimmobilizing a nucleic acid aptamer having a sequence selected from thegroup consisting of the base sequences set forth in SEQ ID NOS: 19 to28, wherein the nucleic acid aptamer is specifically bound to a BovineViral Diarrhea Virus type
 1. 10. The solid phase carrier of claim 9,wherein the solid phase carrier includes a substrate, a resin, a plate,a filter, a cartridge, a column, or a porous material.
 11. The solidphase carrier of claim 10, wherein the substrate includes a nickel-PTFE(polytetrafluoroethylene) substrate, a glass substrate, an apatitesubstrate, a silicon substrate, a gold substrate, a silver substrate, oran alumina substrate.
 12. The solid phase carrier of claim 9, whereinthe solid phase carrier is used to detect or separate the Bovine ViralDiarrhea Virus type
 1. 13. The solid phase carrier of claim 9, whereinthe solid phase carrier is used to diagnose bovine viral diarrhea.
 14. Akit for detecting a Bovine Viral Diarrhea Virus type 1 comprising: anucleic acid aptamer having a sequence selected from the groupconsisting of the base sequences set forth in SEQ ID NOS: 19 to 28,wherein the nucleic acid aptamer is specifically bound to a Bovine ViralDiarrhea Virus type
 1. 15. A kit for diagnosing bovine viral diarrheacomprising: a nucleic acid having a sequence selected from the groupconsisting of the base sequences set forth in SEQ ID NOS: 19 to 28,wherein the nucleic acid aptamer is specifically bound to a Bovine ViralDiarrhea Virus type
 1. 16. A composition for separating a Bovine ViralDiarrhea Virus type 1 comprising: a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 19 to 28, wherein the nucleic acid aptamer isspecifically bound to a Bovine Viral Diarrhea Virus type
 1. 17. A BovineViral Diarrhea Virus type 1 separation method comprising: bringing aBovine Viral Diarrhea Virus type 1-containing sample in contact with asolid phase carrier immobilizing a nucleic acid aptamer having asequence selected from the group consisting of the base sequences setforth in SEQ ID NOS: 19 to 28 in which the nucleic acid aptamer isspecifically bound to a Bovine Viral Diarrhea Virus type 1; and elutingthe Bovine Viral Diarrhea Virus type 1 bound to the solid phase carrierusing an eluent.